PDX1+ cell budding morphogenesis in a stem cell-derived islet spheroid system

Remarkable advances in protocol development have been achieved to manufacture insulin-secreting islets from human pluripotent stem cells (hPSCs). Distinct from current approaches, we devised a tunable strategy to generate islet spheroids enriched for major islet cell types by incorporating PDX1+ cell budding morphogenesis into staged differentiation. In this process that appears to mimic normal islet morphogenesis, the differentiating islet spheroids organize with endocrine cells that are intermingled or arranged in a core-mantle architecture, accompanied with functional heterogeneity. Through in vitro modelling of human pancreas development, we illustrate the importance of PDX1 and the requirement for EphB3/4 signaling in eliciting cell budding morphogenesis. Using this new approach, we model Mitchell-Riley syndrome with RFX6 knockout hPSCs illustrating unexpected morphogenesis defects in the differentiation towards islet cells. The tunable differentiation system and stem cell-derived islet models described in this work may facilitate addressing fundamental questions in islet biology and probing human pancreas diseases.

5. Figure S2B.Looking at flow cytometry data for PDX1 and NKX6.1 it looks like the cell lines tested do not activate NKX6.1 at stage 4? This is in contrast of graph S2D where it looks like there are NKX6.1 postive cells.
6. Can the authors show the staining for the whole cluster of Figure 1E, not only the islet bud? 7. The high enrichment of INS cells in localized bud structures seem to also happen in stage 7 wnt med condition (figure S1A)?They authors should discuss in more details why this new system could be advantageous compared to previous method.8. Several times the authors refer to data not shown.Can they explain why or show the data in supplementary figures?9. Figure 5E: the genes are very difficult to correlate with the lines in the heatmap.The authors can maybe divide the heatmap in different parts so that the names of the genes can be better annotates.
10. Figure 5H: some qPCR gene expression data does not correlate with the RNAseq data showed in heatmap for the same genes.For example KRT18 is showed as not significantly expressed by qPCR, but seem to have a very big difference in gene expression on heatmap between bodies and buds... Could the authors find a better way of plotting qPCR data such as relative gene expression without fold change comparison so that the real gene expression levels can be better visualized?Human islet controls can be plotted next to the experimental conditions if needed.11. Figure 6J does not show a striking change in hormonal cells thereby suggesting that migration of PDX1 cells and budding might not be essential.It would be very useful to demonstrate that Ephrin inhibition increases/decreases the number of multi hormonal cells.This could show that their budding system and the PDX1 migration is indeed necessary to produce functional endocrine cells.
Reviewer #2 (Remarks to the Author): In this manuscript, Kieffer and colleagues developed a strategy for generating stem cell-derived islet spheroids that incorporates a PDX1+ cell budding step in the differentiation protocol.The 'buddingtype differentiation' was achieved by lowering the level of Wnt signaling in culture at the endoderm differentiation stage.The authors showed that this differentiation strategy leads to a core-mantle arrangement of the islet cells, with the β-cells in a core position while α-cells in the mantle, and an enhancement of islet cell maturation.In addition, the EphB3/4 signaling was found to be involved in PDX1+ cell budding morphogenesis.Based on the observations presented in this study, the authors proposed that the PDX1+ budding step mimics the in vivo islet morphogenesis process, whereby PDX1+ progenitor cells would first cluster and subsequently develop into islet cells.This model resembles the way principal islets are formed in lower vertebrates, such as fish and lamprey models.
The question investigated in this paper is interesting and relevant for the islet biology as well as diabetes cell therapy fields.Indeed, learning how to reproduce islet morphogenesis in the differentiation protocols is needed for improving the functional maturity of stem cell-derived islets.However, there are a number of issues that need to be addressed here to support the conclusions drawn by the authors.I listed below specific concerns with the manuscript: 1) The authors made use of the sentence 'data not shown' multiple times in the manuscript(see pp. 3, 6, 7, Results section).This not acceptable, the data should be always included in the Main or Supplementary figures.Especially, some of these 'data not shown' results are particularly relevant being either control experiments (e.g.Wnt target gene modulation should be shown as control of an effective Wnt low/med strategy) or evidence directly in support of major conclusions (e.g.evidence for Robo/ Rho-actin/ Wnt signalings not affecting PDX1+ cell clustering should be shown and not reported as 'data not shown').
2) In Figure 1E, hormone+ cells are not clustered as shown in Fig. 1 C and D with the INS-GFP line.An important control missing here is a co-staining of the INS-GFP-derived clusters with an anti-insulin antibody and anti-GFP to show the overlay between the two signals.
3) The movies performed with the PDX1-reporter hESC line in support of the migration of PDX1+ cells need to be included in the paper.
4) The conclusion drawn from the analysis of the PDX1-KO hESCs phenotype should be revised.It is known that in the absence of PDX1 pancreatic fate specification does not occur; budding morphogenesis cannot start here as there are no pancreatic progenitors in the culture.Thus, the statement ' … expression of PDX1 induces cells to undergo budding morphogenesis in our spheroid system' (see p. 5, Results section) is wrong and needs to be revised.The experiments performed on PDX1-KO line could be entirely removed, as it is inconclusive.Actually, the results obtained with the RFX6-KO line suggest that high /sustained levels of PDX1 are required to initiate the clustering of the pancreatic progenitors and, possibly, subsequent islet morphogenesis.This would be in line with the well-known increase of PDX1 levels in vivo in the trunk / future endocrine cells at later stages after pancreatic specification.Thus, an hypomorphic allele or temporally controlled mutation would be more appropriate here instead of using a PDX1 KO line.
5) The characterization of the two compartments 'islets buds and main bodies' needs to be expanded.Bulk RNASeq and RT-qPCR validation showed a set of differentially expressed genes between the islet buds and main bodies, however, based on both analyses one cannot exclude the presence cell type mixtures in each compartment.For instance, RT-qPCR (see Figs. 5I-K) showed fibroblast markers (THY1, COLA1, VIM) being expressed also in GFP+ islets buds and not exclusively in the main bodies.Thus, one cannot rule out the presence of fibroblasts in the islets bud compartment and their potential role in islet morphogenesis/differentiation and maturation.The same applies to exocrine marker genes.The authors should characterize the main differences between the 2 compartments at least by immunostaining, as they did for the Ephrin signaling molecules (see Fig. S7).Moreover, in this paper the 2 compartments were separated at stage S6, which is quite late in the differentiation protocol; thus, one can only conclude that surrounding cues/cell-cell interactions are not required for stem cell-derived islet maturation but cannot exclude an earlier role in differentiation and morphogenesis.This should be rectified in the manuscript.Alternatively, the experiment should be designed in a different way and includes the separation of the 2 compartments at earlier time points.
6) The analysis of NGN3 expression should be examined upon EphB signaling inhibition and added to complete the analysis in Fig. 6 I,J.
7) The model proposed here is unclear.It is well established that all pancreatic progenitors once specified are PDX1+ cells.Thus, according to their model, PDX1+ cells should all undergo clustering and, subsequently, give rise to islet cells.This would be mean that an entire pancreatic bud would become an islet, which is unlikely.Perhaps, there is a certain heterogeneity among PDX1+ pancreatic progenitors and the difference between a PDX1+ pancreatic progenitor that becomes an islet cell or not lies in the PDX1 levels.This seems a more likely model.Also, the legend of Figure 7 accompanying the model should be revised.The statement '...we observe PDX1+ cell budding morphogenesis prior to pancreatic specification ...' is inaccurate.PDX1 is a transcription factor required for pancreatic fate specification.Perhaps, the authors meant 'prior to endocrine specification'?8) A core-mantle islet architecture is typical of the mouse islets, while in human the islets cells are scattered and intermingled.Based on this study, it seems that the murine core-mantle islet architecture performs better than the human one in a stem cell culture model.The authors should discuss these aspects in the Discussion section.
Reviewer #3 (Remarks to the Author): In this manuscript, Zhao and Liang et al demonstrate a new protocol for hESC modelling of pancreas budding morphogenesis.They demonstrate that Wnt concentrations at the beginning of the differentiation protocol is critical for inducing the PDX1+ dependent budding morphogenesis model and they validate their findings using RNA-Seq of the buds vs main body of the spheroids.They use the model to study the role of PDX1-RFX6 transcription factors in budding human morphogenesis and also demonstrate a role for Ephrin signaling in the budding morphogenesis process.The manuscript is clear, well written and the importance of a human model for embryonic human pancreas budding morphogenesis is of interest to the scientific community.While most of the work is convincing there are certain points that could be further clarified: Major points: -The classification and characterization of the Wntmed and Wntlow should be more clear in the manuscript.The range of the Chir concentrations to separate these two conditions are quite narrow.I would suggest that a range of concentration necessary for bud formation needs to be tested and clearly reported in the figure to make the protocol clear, i.e what is a low concentration of Chir that bud is no longer formed?-How efficient and reproducible is the bud formation during the differentiation process with the new protocol?Please report the percentage of clusters that form a bud during the differentiation.Is there any spontaneous bud formation observed with the Wntmed conditions?-Is there any extracellular matrix coating used during the differentiation?Please include this information as it will be necessary for the reproduction of the protocol -Does Pdx1 directly regulates Ephrin signaling and expression?It would be interesting to check the Pdx1 KO line for expression of Ephrin proteins.
-To support further the model and connect with the literature of budding morphogenesis that the authors discuss, it would be interesting to check how the bud "connects" to the main body.Please stain for adhesion molecules e.g ECadherin-integrin molecules-EMT markers and cell polarity markers to get a more clear insight into the budding process.Minor points: -Assess directly the PDX1 proliferation rate (e.g EdU incorporation and FACS) during bud formation.
-Some of the data not shown parts would be informative to be included for example: 1. Wnt target genes 2. The screen of the individual components of stage 7 media 3.The data on the screen of different signaling inhibitors on page 6 -If feasible in Supplementary figure 7 validate ephrin expression by western blot -For completeness in the zebrafish references please cite this work for ephrin signaling in liver bud formation: PMID: 27825440 and this reference that first showed bud formation in zebrafish larva: PMID: 12941629 -A recent preprint used Rfx6 KO iPSC system in the pancreas context, please discuss in relation to your findings https://doi.org/10.1101/2023.11.15.567202Reviewer #1 (Remarks to the Author): Please find my comments the manuscript en4tled "PDX1+ cell budding morphogenesis in a stem cellderived islet spheroid system" by Jia Zhao and colleagues.This report includes data showing a new protocol to generate pancrea4c islet cells from hPSCs.For that, the authors refine their previous protocol for endoderm differen4a4on by fine tuning Wnt signalling.They observed that low Wnt condi4on allows the produc4on of budding aggregate containing PDX1 cells.These PDX1 progenitors can further differen4ate into endocrine cells including Insulin secre4ng cells.The authors them demonstrate the interest of their system to perform gene func4on analyses using hPSCs knock out for PDX1 and RFX6.Finally, they uncovered that the Ephrin pathway is involved in the bud forma4on and the recruitment of PDX1 cells.This is an interes4ng study and only few aspects need to be bePer detailed: Response: We thank the reviewer for their posi4ve assessment of our work and for the sugges4ons below on how to improve upon our study.We have performed addi4onal experiments and analyses following your sugges4ons.The manuscript text has been extensively revised (highlighted with yellow background), and now contains new data within main figures plus 11 new supplementary figures and supplementary tables.Below please find our detailed point-to-point responses in blue font.
1. How transcriptomically different is the appearance of pancrea4c cells in the Wnt med and Wnt low protocols?Wnt low protocol has less PDX1+ cells, but what differs between both protocols in terms of appearance and matura4on of pancrea4c endocrine and islet cells?Is the morphogenesis/PDX1 rearrangement the 'only' difference?Response: We thank the reviewer for raising these ques4ons.To bePer assess the differences in the cells generated by the Wnt-med and Wnt-low protocols, we have performed the following experiments: (1) RT-qPCR assays to examine a panel of key transcripts at mul4ple differen4a4on stages (Supplementary Fig. 3); and (2) sta4c glucose s4mulated insulin secre4on (GSIS) assays to assess the func4onality of sorted INS+ beta cells at Stage 7 (Supplementary Fig. 1h).We find that pancrea4c cell transcripts (PDX1, NKX6.1, SOX9) and pro-endocrine cell transcripts (NGN3, NEUROD1) were lower in budding type Stage 4 cells generated in Wnt low condi4ons than cells obtained from the Wnt medium bulk-type differen4a4on condi4on (Supplementary Fig. 3b).However, the expression levels of most transcripts became comparable between budding type and bulk type cells at Stages 5 and 7 (Supplementary Fig. 3c-3d).Nevertheless, rela4ve to bulk type cells, we no4ce higher levels of GHRL, SLC18A1, KCNK1 transcripts and lower levels of GCG, PPY, ARX, NKX2.2, ABCC8 transcripts in the budding type Stage 7 cells (Supplementary Fig. 3d).Although transcripts of func4onal beta cell markers MAFA, IAPP, GCK, PSCK1 and KCNK3 were similar between the two types of differen4a4on (Supplementary Fig. 3d), insulin secre4on and total insulin content of budding type Stage 7 beta cells was 1.4-fold and 1.7-fold lower than those of bulk type cells, respec4vely (Supplementary Fig. 1h), sugges4ng a less mature phenotype of beta cells generated by the budding protocol.To summarize these findings, we also created a new supplementary table that highlights key differences of the cells generated by the two protocols (Supplementary Table 1).We have included these new results in the revised manuscript (Page 3).
2. Figure S1B.The authors report around 5% of PDX1/NKX6.1 cells in stage 4 (text and b flow cytometry), however, on the immunostaining showed on this figure for stage 4, NKX6.1 posi4ve cell are difficult to find.Can the authors provide a bePer staining?Response: Thank you for this sugges4on.We have now replaced this image with a more representa4ve immunostaining image that shows the detec4on of a few NKX6.1-posi4ve cells in the budding type stage 4 cells (Supplementary Fig. 1e).
4. Similarly, the authors claim that their protocol can work on mul4ple hPSCs.However, there is liPle informa4on about efficacy of bud forma4on.Can they show staining showing bud forma4on with each cell line and provide the efficiency of bud forma4on.Response: Please see our detailed responses above to Comment #3.Our new data reveal high efficiency and reproducibility of bud forma4on observed with the Wnt low protocol in various stem cell lines (Supplementary Fig. 5 and Pages 3-4).Representa4ve immunostaining and live cell images showing bud forma4on from each cell line are included in the revised manuscript (Fig. 1c and Supplementary Figs.4e-4f, 5 and 11).
5. Figure S2B.Looking at flow cytometry data for PDX1 and NKX6.1 it looks like the cell lines do not ac4vate NKX6.1 at stage 4? This is in contrast of graph S2D where it looks like there are NKX6.1 posi4ve cells.Response: In Figure S2B (now Supplementary Fig. 4b, representa4ve flow cytometry data) and Figure S2D (now Supplementary Fig. 4d, the quan4fica4on), we show that NKX6.1+ cells can be induced at stage 4 but at extremely low percentages.On average, 0.5%-3% NKX6.1+ cells are detected at stage 4 in the cell lines we tested.
6. Can the authors show the staining for the whole cluster of Figure 1E, not only the islet bud?Response: Indeed, Figure 1E shows immunostaining of an isolated stage 7 islet bud, to highlight that the islet bud compartment contains major islet cell types.Nevertheless, as requested we have now added a supplementary figure (Supplementary Fig. 6) with several examples showing the staining for whole clusters with both compartments of islet bud and main body.
7. The high enrichment of INS cells in localized bud structures seem to also happen in stage 7 wnt med condi4on (figure S1A)?They authors should discuss in more details why this new system could be advantageous compared to previous method.Response: Local enrichment of INS+ cells may be seen in stage 7 Wnt-med condi4ons, which is in line with similar observa4ons in other publica4ons (PMID: 35241836 and PMID: 34380694).This may represent the process of islet cell rearrangement at maturing stages in developing human islets.By contrast, our budding model emphasizes the budding morphogenesis of PDX1+ progenitor cells, recapitula4ng pancrea4c bud forma4on, and an event occurring at an earlier developmental stage.
Compared to previous methods, this new system provides a unique human islet developmental model to study asynchronous differen4a4on within a heterogeneous cell-cell interac4on environment, whereby pancrea4c cell sor4ng, 4ssue segrega4on and islet morphogenesis occur.This approach may complement the use of human fetal pancreas 4ssues, mouse organoid models and other tools using rodents.We now men4on these points in the Discussion (Page 8) and have included a new summary table (Supplementary Table 1) highligh4ng key differences and u4lity/advantages of this new system compared to previous methods.
8. Several 4mes the authors refer to data not shown.Can they explain why or show the data in supplementary figures?Response: Thank you for raising this concern.We have now added all these data in new supplementary figures to improve the transparency of our results.These include: (1) qPCR 96-well plate array data showing differen4al Wnt pathway genes by Wnt-med and Wnt-low condi4ons (Supplementary Fig. 2); (2) immunostaining data showing the screen of individual components of stage 7 media on islet architecture (Supplementary Fig. 9); and (3) live cell fluorescent imaging data showing the screen of different signaling pathways on PDX1+ cell clustering (Supplementary Fig. 14).9. Figure 5E: the genes are very difficult to correlate with the lines in the heatmap.The authors can maybe divide the heatmap in different parts so that the names of the genes can be bePer annotates.Response: Thank you for this helpful sugges4on to improve clarity.We have created a new heatmap highligh4ng the selec4ve genes of interests in Fig. 5f to improve data visualiza4on such that the names of genes and the varied expression of a par4cular gene across different samples are now bePer annotated.
10. Figure 5H: some qPCR gene expression data does not correlate with the RNA-seq data showed in heatmap for the same genes.For example, KRT18 is showed as not significantly expressed by qPCR, but seem to have a very big difference in gene expression on heatmap between bodies and buds... Could the authors find a bePer way of plowng qPCR data such as rela4ve gene expression without fold change comparison so that the real gene expression levels can be bePer visualized?Human islet controls can be ploPed next to the experimental condi4ons if needed.Response: We thank this reviewer for poin4ng out the differences in qPCR and RNA-seq plots.The differences in RNA-seq heatmap and qPCR plots are partly due to the different techniques and normaliza4on methods.In RNA-seq, gene counts axer DESeq2 normaliza4on and variance stabilizing transforma4on (~ log2 scale) are converted to row z-scores to bePer illustrate the differences across samples on heatmap.qPCR data were first normalized to a housekeeping gene (NFX1), and then the rela4ve gene expression was compared to the expression level in human islets.In the case of KRT18, the RNA-seq data (~log2 scale) of body and bud are 8.84 ± 0.06 versus 8.27 ± 0.10 (t-test p = 0.01), and their z-scores (axer row z-score scaling) makes the difference look more striking on heatmaps.The expressions of KRT18 in qPCR are 0.32 ± 0.09 in body and 0.15 ± 0.03 in bud (t-test p = 0.07).Consistently, both techniques show that body cells tend to express higher level of KRT18 transcripts rela4ve to bud cells.
We agree with this reviewer that rela4ve gene expression levels are more informa4ve than fold change comparison.Following this sugges4on, we have ploPed our qPCR data as gene expression levels rela4ve to human islets in the revised Fig. 5i-5l.
11. Figure 6J does not show a striking change in hormonal cells thereby sugges4ng that migra4on of PDX1 cells and budding might not be essen4al.It would be very useful to demonstrate that Ephrin inhibi4on increases/decreases the number of mul4 hormonal cells.This could show that their budding system and the PDX1 migra4on is indeed necessary to produce func4onal endocrine cells.Response: The Figure 6J (now is Fig. 6k) showed sta4s4cally significant changes in the percentages of hormonal cells axer EphB inhibi4on (INS+ cells: 43% ± 2.6% in DMSO versus 19.5% ± 3.7% in EphB3/4 co-inhibi4on; and GCG+ cells: 18.7% ± 1.2% in DMSO versus 8.1% ± 1.6% in EphB3/4 co-inhibi4on).As per sugges4on from Reviewer #2, we also added new data in Figure 6I showing that NGN3 expression was significantly reduced axer EphB inhibi4on (6.7% ± 0.8% in DMSO versus 1.7% ± 0.4% in EphB3/4 co-inhibi4on).Collec4vely, these results support our conclusion that PDX1 cell clustering is an important step to produce subsequent endocrine precursors and islet cells (Fig. 6).We have now clarified these results in the revised manuscript (Page 8).Nevertheless, we are aware that the EphB3/4 co-inhibi4on treatment may not completely block EphB signaling (Supplementary Figs. 14 and 16), and we have discussed this limita4on in our revised manuscript (Page 9).

Reviewer #2 (Remarks to the Author):
In this manuscript, Kieffer and colleagues developed a strategy for genera4ng stem cell-derived islet spheroids that incorporates a PDX1+ cell budding step in the differen4a4on protocol.The 'budding-type differen4a4on' was achieved by lowering the level of Wnt signaling in culture at the endoderm differen4a4on stage.The authors showed that this differen4a4on strategy leads to a core-mantle arrangement of the islet cells, with the β-cells in a core posi4on while α-cells in the mantle, and an enhancement of islet cell matura4on.In addi4on, the EphB3/4 signaling was found to be involved in PDX1+ cell budding morphogenesis.Based on the observa4ons presented in this study, the authors proposed that the PDX1+ budding step mimics the in vivo islet morphogenesis process, whereby PDX1+ progenitor cells would first cluster and subsequently develop into islet cells.This model resembles the way principal islets are formed in lower vertebrates, such as fish and lamprey models.Response: We thank this reviewer for their 4me assessing our manuscript and for the though|ul summary of our findings.
The ques4on inves4gated in this paper is interes4ng and relevant for the islet biology as well as diabetes cell therapy fields.Indeed, learning how to reproduce islet morphogenesis in the differen4a4on protocols is needed for improving the func4onal maturity of stem cell-derived islets.However, there are a number of issues that need to be addressed here to support the conclusions drawn by the authors.I listed below specific concerns with the manuscript: Response: We appreciate the suppor4ve comments from this reviewer.We have now addressed these concerns by performing addi4onal experiments and adding new data, analysis, and relevant discussions.
The manuscript text has been extensively revised (highlighted with yellow background), and now contains new data within main figures plus 11 new supplementary figures and supplementary tables.Point-to-point responses are detailed below to address each specific concern.
1) The authors made use of the sentence 'data not shown' mul4ple 4mes in the manuscript (see pp. 3, 6, 7, Results sec4on).This not acceptable, the data should be always included in the Main or Supplementary figures.Especially, some of these 'data not shown' results are par4cularly relevant being either control experiments (e.g., Wnt target gene modula4on should be shown as control of an effec4ve Wnt low/med strategy) or evidence directly in support of major conclusions (e.g., evidence for Robo/ Rho-ac4n/ Wnt signaling not affec4ng PDX1+ cell clustering should be shown and not reported as 'data not shown').Response: We thank the reviewer for raising this concern.We have now added all these data in new supplementary figures to improve the transparency of our results.These include: (1) qPCR 96-well plate array data showing differen4al Wnt pathway genes by Wnt-med and Wnt-low condi4ons (Supplementary Fig. 2); (2) immunostaining data showing the screen of individual components of stage 7 media on islet architecture (Supplementary Fig. 9); and (3) live cell fluorescent imaging data showing the screen of different signaling pathways on PDX1+ cell clustering (Supplementary Fig. 14).
2) In Figure 1E, hormone+ cells are not clustered as shown in Fig. 1 C and D with the INS-GFP line.An important control missing here is a co-staining of the INS-GFP-derived clusters with an an4-insulin an4body and an4-GFP to show the overlay between the two signals.

Response:
We are sorry for the lack of clarity here.Figure 1E shows immunostaining of an isolated stage 7 islet bud and it intends to highlight that the islet bud compartment contains the major islet cell types.We have now added a supplementary figure (Supplementary Fig. 6) with several examples showing the staining for whole clusters with both compartments of islet bud and main body.
We agree with the reviewer that it is important to demonstrate the reliability of the INS-GFP reporter line that we used in this study.We have performed both immunostaining and flow cytometry analysis, showing 87.1% (from n = 2 independent experiments) overlay of GFP signals by an4-GFP an4body and insulin signals by an4-insulin an4body (Supplementary Fig. 19a-19c).Similarly, the PDX1 reporter line (HUES4 PDXeG) was validated by imaging GFP reporter signals and PDX1 signals by an4-PDX1 an4body, illustra4ng 89.1% (from n = 2 independent experiments) overlay of signals (Supplementary Fig. 19d-19f).We have added these data and specified this informa4on in the revised manuscript (Page 12).
3) The movies performed with the PDX1-reporter hESC line in support of the migra4on of PDX1+ cells need to be included in the paper.Response: We apologize for giving the readers the impression that 4me-lapse movies were generated.Rather, these data obtained with the PDX1 reporter hESC line are 4me-course snapshot images (Figs.2d, 6d and Supplementary Figs. 5, 8) rather than 4melapse images.To track the process of PDX1+ cell migra4on in living cultures, we differen4ated individual clusters in ultralow aPachment U-boPom 96-wells and captured several images from each well with a conven4onal fluorescent microscope on a daily basis.We have clarified this informa4on in the revised manuscript (Page 4).Although we do not have a suitable imager capable of doing long-term 4melapse imaging, we feel that our findings s4ll support our conclusion of PDX1+ cell migra4on and clustering.
4) The conclusion drawn from the analysis of the PDX1-KO hESCs phenotype should be revised.It is known that in the absence of PDX1 pancrea4c fate specifica4on does not occur; budding morphogenesis cannot start here as there are no pancrea4c progenitors in the culture.Thus, the statement '… expression of PDX1 induces cells to undergo budding morphogenesis in our spheroid system' (see p. 5, Results sec4on) is wrong and needs to be revised.The experiments performed on PDX1-KO line could be en4rely removed, as it is inconclusive.Actually, the results obtained with the RFX6-KO line suggest that high /sustained levels of PDX1 are required to ini4ate the clustering of the pancrea4c progenitors and, possibly, subsequent islet morphogenesis.This would be in line with the well-known increase of PDX1 levels in vivo in the trunk / future endocrine cells at later stages axer pancrea4c specifica4on.Thus, an hypomorphic allele or temporally controlled muta4on would be more appropriate here instead of using a PDX1 KO line.Response: Thank you for these construc4ve comments.We fully acknowledge that since knockout of PDX1 disrupts the forma4on of the pancrea4c progenitor pool, it is not possible to determine the role of PDX1 in downstream events from analysis of PDX1 knockout cells.We now ensure that in the revised manuscript, we separate our conclusion regarding the role of PDX1 in budding morphogenesis from studies with the PDX1 knockout cells.Specifically, we have removed the concluding sentence "expression of PDX1 induces cells to undergo budding morphogenesis in our spheroid system" in this result sec4on, and changed the concluding remarks to "These results corroborate previous work by recapitula4ng the pancrea4c agenesis phenotype with a stem cell model and again emphasize the importance of PDX1 in pancrea4c fate specifica4on and morphogenesis" in the revised manuscript.
We rely on several other observa4ons to support the role of PDX1 in budding morphogenesis, including as the reviewer notes, our interes4ng findings with the RFX6-KO line demonstra4ng that the sustained expression of PDX1 is required to ini4ate the clustering of pancrea4c progenitors and subsequent islet cell morphogenesis in a heterogeneous cellular environment (i.e., containing both PDX1-expressing cells and PDX1-nega4ve cells).Our conclusion is also supported by our live cell tracking experiments in PDX1 reporter hESC cultures (Fig. 2d and Supplementary Fig. 8a-8i).Specifically, with the use of a hypomorphic PDX1-EGFP reporter line (i.e., one PDX1 allele is replaced by EGFP sequence), we provided direct evidence that pancrea4c budding morphogenesis is mediated by spontaneous clustering of PDX1+ cells.While experiments performed with the PDX1 KO line cannot inform the role of PDX1 beyond pancrea4c progenitor forma4on, the studies s4ll illustrate the u4lity of our stem cell systems in modelling human pancreas diseases.
5) The characteriza4on of the two compartments 'islets buds and main bodies' needs to be expanded.Bulk RNA-Seq and RT-qPCR valida4on showed a set of differen4ally expressed genes between the islet buds and main bodies, however, based on both analyses one cannot exclude the presence cell type mixtures in each compartment.For instance, RT-qPCR (see Figs. 5I-K) showed fibroblast markers (THY1, COLA1, VIM) being expressed also in GFP+ islets buds and not exclusively in the main bodies.Thus, one cannot rule out the presence of fibroblasts in the islets bud compartment and their poten4al role in islet morphogenesis/differen4a4on and matura4on.The same applies to exocrine marker genes.The authors should characterize the main differences between the 2 compartments at least by immunostaining, as they did for the Ephrin signaling molecules (see Fig. S7).Moreover, in this paper the 2 compartments were separated at stage S6, which is quite late in the differen4a4on protocol; thus, one can only conclude that surrounding cues/cell-cell interac4ons are not required for stem cell-derived islet matura4on but cannot exclude an earlier role in differen4a4on and morphogenesis.This should be rec4fied in the manuscript.Alterna4vely, the experiment should be designed in a different way and includes the separa4on of the 2 compartments at earlier 4me points.Response: Thank you for these construc4ve comments.Guided by our own bulk RNA-seq and qPCR data, we have now immunostained several differen4ally expressed candidate genes, including THY1, VIM, SOX9, YAP1 and SOX2 (an anterior foregut marker), to further characterize main body cells as per the sugges4on (Supplementary Fig. 13).Specifically, we show (1) remarkably higher expression levels of THY1 and VIM (pancrea4c mesenchyme or fibroblast markers) in the main bodies; (2) mutually exclusive expression of the ductal cell marker SOX9 in main body cells and the endocrine cell marker NEUROD1 in bud cells; (3) higher expression level of YAP1 (a mechanosensi4ve signal for balancing progenitor cell self-renewal and differen4a4on) in the main body cells versus lower expression of YAP1 in the NEUROD1+ endocrine-commiPed bud cells; and (4) higher expression of the anterior foregut marker SOX2 in main body cells.We also examined the exocrine cell marker Trypsin 1/2/3 but did not detect any expression in either bud or body cells (Supplementary Fig. 13), ruling out the exocrine iden4ty of main body cells.Collec4vely, these results suggest a poten4al mesenchyme/fibroblast phenotype or other lineage commitment (ductal or anterior foregut) but not an exocrine lineage of the main body cells.We have now added these results in the revised manuscript (Supplementary Fig. 13 and Page 7).
We agree with this reviewer that we cannot exclude an earlier role of surrounding cues from main body cells in islet bud morphogenesis/differen4a4on.Due to technical limita4on, we are only able to separate the two compartments at early stage 6 (e.g., S6D3) when the buds are more protruding from main bodies.We have discussed this limita4on in our revised manuscript (Pages 7 and 11).
6) The analysis of NGN3 expression should be examined upon EphB signaling inhibi4on and added to complete the analysis in Fig. 6 I, J. Response: Thank you for this sugges4on.Although we are s4ll working on protocol op4miza4on for assaying NGN3 expression under flow cytometry, we have successfully examined NGN3 expression by immunostaining to complete the analysis in Figure 6I-6J (now is Figure 6J-6K).By normaliza4on to the total PDX1+ cells, we show that NGN3 expression is significantly impaired upon EphB3/4 co-inhibi4on compared to DMSO control (1.7% ± 0.4% in EphB3/4 co-inhibi4on versus 6.7% ± 0.8% in DMSO).We have added these data in the new Fig.6i and discussed these results in the revised manuscript (Page 8).
7) The model proposed here is unclear.It is well established that all pancrea4c progenitors once specified are PDX1+ cells.Thus, according to their model, PDX1+ cells should all undergo clustering and, subsequently, give rise to islet cells.This would be mean that an en4re pancrea4c bud would become an islet, which is unlikely.Perhaps, there is a certain heterogeneity among PDX1+ pancrea4c progenitors and the difference between a PDX1+ pancrea4c progenitor that becomes an islet cell or not lies in the PDX1 levels.This seems a more likely model.Also, the legend of Figure 7 accompanying the model should be revised.The statement '...we observe PDX1+ cell budding morphogenesis prior to pancrea4c specifica4on ...' is inaccurate.PDX1 is a transcrip4on factor required for pancrea4c fate specifica4on.Perhaps, the authors meant 'prior to endocrine specifica4on'?Response: Thank you for these construc4ve comments.We demonstrate that a vast majority of PDX1+ cells undergo clustering and budding out from PDX1-nega4ve main bodies prior to endocrine specifica4on (Fig. 2b, 2d and Supplementary Figs. 5, 8, 15) in our budding type differen4a4on system.With our media highly enriched with endocrine cell-directed cocktails (which inhibits progenitor cell prolifera4on and does not support ductal or exocrine cell commitment) at later stages, the PDX1+ pancrea4c bud efficiently gives rise to an endocrine islet (Fig. 2a).This paradigm is supported by observa4ons of islet forma4on in fish and lamprey, where a dorsal bud is formed through the convergence of PDX1+ cells that later develop into a principal islet (PMIDs: 30133710, 22034951, 11377827 and 26435359).Nevertheless, we note that only a certain popula4on of PDX1+ progenitor cells, very likely the ones with high/peak PDX1 expression level (Fig. 2g), are specified to islet cells within the bud structure (Figs.2a, 2f-2h, 4e), in line with this reviewer's comments.We do not wish to give readers the impression that all PDX1+ cells become islet cells.The model proposed in the original schema4c of Fig. 7a intended to show that the bud structure formed by PDX1+ cell clustering creates a favorable niche and subsequently promotes local pro-endocrine cell and islet cell commitment.We have now revised Fig. 7a to show the par4al endocrine commitment and made further clarifica4on in the legend of Fig. 7 (Page 20).We also have corrected "prior to pancrea4c specifica4on" to "prior to endocrine specifica4on" in our revised manuscript.8) A core-mantle islet architecture is typical of the mouse islets, while in human the islets cells are scaPered and intermingled.Based on this study, it seems that the murine core-mantle islet architecture performs bePer than the human one in a stem cell culture model.The authors should discuss these aspects in the Discussion sec4on.Response: We thank the reviewer for raising this important observa4on.In our budding type differen4a4on model, the fact that stage 7 islet buds display bePer func4onality than stage 6 islet buds may emphasize that both architecture and cellular maturity states (e.g., mono-or bi-hormonal) determine islet func4on.Indeed, we show the majority of stage 6 islet cells are INS+/GCG+ bi-hormonal cells adop4ng an intermingled structure, whereas stage 7 islet cells are mostly INS+/GCG-and INS-/GCG+ monohormonal cells with a favorable core-mantle organiza4on (Fig. 3a-3c).Moreover, the core-mantle architecture renders beta cells with more homotypic cell-cell interac4ons, which is associated with more synchronized calcium ac4vi4es under glucose s4mula4on (Fig. 3e-3g), providing a plausible explana4on that the coremantle islet architecture performs bePer than a mixed one in our stem cell model.In addi4on, the Susan Bonner-Weir group reported that small-sized human islets prefer an adop4on of core-mantle paPern (like mouse islets) and large-sized islets with a mixed organiza4on seem derived from small "core-mantle modular units" coalescing (PMID: 25604813), sugges4ng that cellular rearrangement occurs in human islets.We have incorporated this discussion into our revised manuscript (Page 10).
respec4vely, showing > 75% main bodies with only one bud and ~ 10% main bodies with two or more buds (Supplementary Fig. 5c, 5g).Collec4vely, these results demonstrate the high efficiency and reproducibility of bud forma4on observed with the Wnt low protocol in various stem cell lines.We have now added these results in the revised manuscript (Supplementary Fig. 5 and Pages 3-4).
Due to the overall high percentages of PDX1+ cells (normally 95%-99%) in bulk type clusters, we do not see spontaneous PDX1+ bud forma4on observed in Wnt med condi4ons.
-Is there any extracellular matrix coa4ng used during the differen4a4on?Please include this informa4on as it will be necessary for the reproduc4on of the protocol.Response: Thank you for this sugges4on.We apply extracellular matrix (ECM) coa4ng for maintenance culture and planar culture during the first four stages of differen4a4on.Bud induc4on and subsequent endocrine cell differen4a4on during the last three stages are conducted under sta4c suspension culture without ECM coa4ng or moun4ng.We have now emphasized this informa4on in the Methods sec4on in the revised manuscript (Page 12).
-Does Pdx1 directly regulate Ephrin signaling and expression?It would be interes4ng to check the Pdx1 KO line for expression of Ephrin proteins.Response: Thank you for raising this ques4on.In our budding type Stage 5 clusters, the observa4on that PDX1+ cells tend to have lower levels of Ephrin proteins (par4cularly EphrinB2 and EphrinB3) (Fig. 5 and Supplementary Fig. 15) may indicate an associa4on between PDX1 and Ephrin expression.Following this sugges4on, we have examined the expression of Eph and Ephrin in the PDX1 KO line.We find that all five Eph receptors and three Ephrin ligands are expressed in PDX1 KO Stage 5 clusters (Supplementary Fig. 17).However, compared to the differen4al localiza4on of Eph/Ephrin in endocrine buds and main bodies derived from wildtype cell line (Supplementary Fig. 15), we note the differen4al or polarized expression paPern is diminished in the PDX1 KO clusters (Supplementary Fig. 17), sugges4ng that spa4al expression paPern of Eph/Ephrin proteins is associated with the presence or absence of PDX1 and thus the budding morphology.Nevertheless, it requires further in-depth inves4ga4on as to whether Eph or Ephrin signaling and expression is directly regulated by PDX1, which could be an interes4ng work in follow-up studies.We have discussed this result in the revised manuscript (Page 9).
-To support further the model and connect with the literature of budding morphogenesis that the authors discuss, it would be interes4ng to check how the bud "connects" to the main body.Please stain for adhesion molecules e.g., ECadherin-integrin molecules-EMT markers and cell polarity markers to get a much clear insight into the budding process.Response: Thank you for raising this interes4ng ques4on.Following this sugges4on, we have examined adhesion molecules, EMT markers and cell polarity markers in our developing cell aggregates.These include E-Cadherin, integrin (ITGA1), beta-catenin, collagen (COL4A1/A2), ZO-1 (marking 4ght junc4ons), N-Cadherin, Vimen4n and THY1.We found strikingly elevated expression of adhesion molecules E-Cadherin and beta-catenin in the NEUROD1+ endocrine buds (Supplementary Fig. 18), indica4ng an "epithelial" phenotype of bud cells and 4ght cell-cell contacts via adheren junc4ons within the bud compartment.COL4A1/A2, a major component of intra-islet basement membrane proteins, was only expressed in endocrine buds (Supplementary Fig. 18), which is in line with its abundant presence in human islets (PMID: 32894318).By contrast, higher levels of "mesenchymal" and fibroblast phenotype markers Vimen4n and THY1 were expressed in the main bodies (Supplementary Fig. 13), again revealing different cell types of buds and main bodies.The expression of EMT markers led us to hypothesize whether PDX1+ cell budding morphogenesis could be mediated by an EMT mechanism.However, our prior experiments of screening various pathways revealed that the EMT-related TGFβ signaling does not affect PDX1+ cell func4on protein-trunca4ng variant (PTV) -p.His293LeufsTer7.The lack of RFX6 in their homozygous RFX6 KO cells leads to a significant reduc4on in PDX1 transcript at mRNA level, reduced induc4on of PDX1+/NKX6.1+ pancrea4c progenitor cells and CHGA+ pro-endocrine cells as well as fails to generate major islet cell types (INS+ cells and GCG+ cells), similar to our observa4ons using a RFX6 complete gene knockout system (Fig. 4a-4f).Nevertheless, our study reports some unique observa4ons compared to this preprint paper.These include: (1) iden4fica4on of PDX1+ cell clustering and budding morphogenesis at early progenitor specifica4on stage, which is not reported in this preprint study; (2) normal presence of PPY+ cells in islet clusters derived from our RFX6 gene complete KO cells, whereas the preprint study shows a lack of PPY+ cells in the implants in vivo (but the authors observed an upregulated PPY transcript at mRNA level in vitro).We speculate that different RFX6 disrup4on systems and culture formats used by the two studies may contribute to the differences in these independent studies.We have now discussed this preprint paper in rela4on to our findings in our revised manuscript (Page 10).